In order to achieve true color vision, animals must have the ability to compare the outputs of photoreceptors with different wavelengths of absorption. The compound eye of Drosophila is made up of approximately 800 repeating units called ommatidia and each ommatidia is sub-divided into 20 specialized cells. Eight of these cells are photoreceptors required for absorbing light. The six outer photoreceptors (R1-R6) express the wide- spectrum rhodopsin Rh1 and are required for motion detection. These are similar to human rod cells. The two inner photoreceptors in the Drosophila eye (R7 and R8), which are similar to human cone cells, are required for color vision and lie in the same optic path where R7 sits distally on top of the proximal R8 cell. They contain distinct rhodopsin photopigments and their outputs are compared in the medulla part of the optic lobe where each projects their axons. Two distinct classes of ommatidia are stochastically distributed throughout the fly retina: In 70% of ommatidia (yellow (y) ommatidia), R7 contains UV-Rh4 while R8 contains green-Rh6, specializing them to discriminate longer wavelengths of light. In the remaining 30% pale (p) ommatidia, R7 contains UV-sensitive Rh3 while R8 contains blue-Rh5 making these ommatidia well adapted to discriminate among short wavelengths of light. Our lab has recently found that the stochastic choice to become a y or p ommatidium is made in the distally located R7 cell, when the transcription factor Spineless is activated. This occurs in 70% of R7 during pupation, specifying them as yR7. The remaining R7 become pR7 by default and subsequently instruct their underlying R8 to become pR8. Here I propose three aims designed to uncover the signaling mechanism required for R8 subtype specification, which leads to robust expression of Rh5 in pR8 and Rh6 in yR8. Recent work in our lab has suggested a role for Dpp signaling, initiated in R7, through the Activin receptor, Babo (in R8) in a non-canonical pathway to establish R8 fate. Babo may then interact with the warts/melted bi-stable loop to maintain this fate. I will examine the entire Dpp and Activin signaling pathways, neither of which have been fully examined for additional or redundant roles in this fate decision. As there are likely additional factors involved in subtype specification, I will also conduct an RNAi-based screen of over 3000 known and predicted membrane- associated and signaling molecules as well as perform subtype expression profiling of p and y R7 and R8 cells using RNAseq techniques. This work will expand our knowledge of retinal patterning and cell fate establishment, specifically following a stochastic decision. It will also add to our knowledge of the Hippo tumor suppressor pathway as well as Dpp and Activin signaling pathways, all of which are utilized in non-canonical ways here. ! PUBLIC HEALTH RELEVANCE: The exclusive expression of specific rhodopsins in the photoreceptors of the Drosophila ommatidia is crucial for correct discrimination and processing of visual cues. The inner photoreceptors (R7 and R8) are divided into two subtypes (pale (p) and yellow (y)), which are stochastically distributed across the compound eye and required for color vision. This proposal aims to characterize the subtype-specific signaling mechanisms initiating in pR7 (Rh3) or yR7 (Rh4) that are required to specify pR8 (Rh5) or yR8 (Rh6) respectively.